{"id":1543,"date":"2017-08-31T19:13:17","date_gmt":"2017-08-31T19:13:17","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?p=1543"},"modified":"2023-03-06T18:00:15","modified_gmt":"2023-03-06T18:00:15","slug":"vitro-testing-anti-viral-activity-flavivirus","status":"publish","type":"post","link":"https:\/\/www.mybiosource.com\/learn\/vitro-testing-anti-viral-activity-flavivirus\/","title":{"rendered":"In Vitro testing for anti-viral activity of flavivirus"},"content":{"rendered":"

Table of Contents<\/strong><\/span><\/h2>\n

I. Introduction<\/strong>
\n\u2022 Dengue as a vector borne disease caused by a flavivirus
\n\u2022 Prevalence of Dengue in tropical and subtropical regions
\nII. Transmission and Life Cycle of Flavivirus<\/strong>
\n\u2022 Vector transmission through mosquitoes
\n\u2022 Attraction of mosquitoes by volatile organic compounds
\n\u2022 Four main serotypes of dengue virus
\n\u2022 Replication of virus in liver and destruction of platelets
\nIII. Dengue Management<\/strong>
\nIV. Screening for Antiviral Activity<\/strong>
\nV. ELISA Test<\/strong>
\nVI. Foci Forming Immunodetection Assay<\/strong>
\nVII. In Situ ELISA Assay<\/strong>
\n\u2022 Addition of primary and secondary antibodies
\n\u2022 Inoculation of color developing solution
\n\u2022 Assay validation<\/strong>
\nVIII. Conclusion<\/strong><\/p>\n

\n

Dengue<\/span> is a vector<\/span> borne disease caused by a flavivirus<\/span><\/em> and is prevalent in both tropical and subtropical regions of the globe and poses a serious challenge to the health fraternity. The lack of an effective antiviral in the market possesses a serious challenge to the healthcare professionals in the management of dengue<\/span> and this leads to the loss of around 25 thousand lives to this disease annually with the numbers increasing every year. Like all viruses, the flavivirus<\/span> <\/em>requires a host for its replication & survival and it is able to transmit the disease to humans by using the mosquitoes, aedes aegypti<\/span> <\/em>as a vector<\/span>. Dengue<\/span> is one of the most prevalent disease caused by mosquitoes. In order to continue its life cycle the flavivirus<\/span><\/em> needs to be transported using a vector<\/span>, recent research findings revealed that during the infectious stage in humans, the flavivirus<\/span><\/em> causes the infected patients skin to release certain volatile organic compounds<\/span> which further attracts more mosquitoes and makes the patient prone to mosquito bites, now these mosquitoes will further act as carrier and will infect the more people leading to a pandemic in the region. There are four main serotypes<\/span> of dengue<\/span> virus, they are DENVI, DENV2, DENV3 and DENV4 with some being specific to particular a region of the globe. When a mosquito (aedes aegypti<\/span>) <\/em>carrying the virus bites a human being, the virus enters the blood stream and starts to replicate in the liver for a period of 2-5 days and results in the lysis of hepatocytes<\/span> releasing more virus which will further infect more cell and multiply. During this phase the virus destroys the platelets resulting in thrombocytopenia<\/span>, a condition where the platelets are below the normal level of 1,50,000\/cu mm. With less platelets the body is unable to stop internal bleedings resulting in hemorrhagic<\/span> fever and will lead to death of the patient. Current treatment options to increase the platelet<\/span> levels are thrombopoietin hormone<\/span> mimetic, platelet<\/span> transfusion and carica papaya leaf extracts. Platelet<\/span> transfusion is usually performed as the last option as in some cases the platelet<\/span> transfusion can cause fluid buildup and this itself can lead to the death of the patient. Some of the complication that arises due to dengue<\/span> is severe dehydration<\/span>, hemorrhagic<\/span> fever, bleeding and dengue<\/span> shock. Dengue<\/span> management requires a comprehensive approach that ensures to maintain the platelet<\/span> level at a level of minimum 80,000 \/cu mm, keeping the temperature down and preventing dehydration<\/span>.<\/p>\n

Although various molecules have exhibited antiviral activity to the flavivirus<\/span><\/em> in invitro studies, most of these studies have not been effective when scaled up as these molecules do not exhibit the same virulence<\/span> invivo. Also, there have be no clinical trials<\/span> reported to have been initiated for any antiviral activity of dengue<\/span> so far.\u00a0 Considering the current scenario the global pandemic threat posed by this viral disease is a major cause of concern and more research is required to screen for an effective antiviral drug. Various phyto chemicals and algal biocompounds have exhibited potential antiviral activity for all four serotypes<\/span> of dengue<\/span> virus. There are various methods that have been established to screen for antiviral activity, each has its own advantages and limitation. The main limitation of these methods are time in sample collection, transportation and sample pre-preparation. The immunodetection technique using ELISA<\/span> can be performed on the site to test for these antiviral activity saving time and allowing more potential samples to be screened for its antiviral activity.<\/p>\n

ELISA<\/span>, is defines as enzyme-linked immunosorbent assay and also called as EIA, it is a test that detects and measures dengue<\/span> antibodies like NS1<\/span>, IgG<\/span> and IgM<\/span> in one\u2019s blood. These antibodies are proteins that the body produces in response to antigens. The ELISA<\/span> test can also be used to determine if one is infected by dengue<\/span> or not. By making certain changes, ELISA<\/span> can also be performed to check for antiviral activity. The main principle behind ELISA<\/span> is antigen<\/span> bind to only specific antibody, the ELISA<\/span> kit contains various small walls which is coated with the antibody of interest and once the sample is added the antigen<\/span> and antibody bind to form a complex. This complex is then viewed by adding specific enzymes which gives a notable color change to the wells where the antibody and antigen<\/span> have bound. The formation of the color complex will be the indicator for the presence of the antigen<\/span> in the sample.<\/p>\n

In case of checking antiviral activity for dengue<\/span> virus, IC50<\/span> (concentration that inhibits 50% of virus infection) and a control must be first prepared. Following which a foci forming immunodetection assay needs to be prepared and for this hepatocytes<\/span> from human source are inoculated and provided with its particular nutrient. \u00a0The cell culture is placed inside the wells of the ELISA<\/span> kit. To the same well flavivirus<\/span> <\/em>group specific monoclonal antibody<\/span> 4g2 is added and immunostaining is performed using a solution of NBT (nitroblue tetrazolium chloride) which is added for color development and detection purpose. The number of wells where antigen<\/span> and antibody has bound will give significant color change, this is counted and expressed as focci forming units. After preparing the focci forming immunodetection, the in situ<\/span> ELISA<\/span> assay is prepared by adding two antibodies, a primary antibody 4g2 and \u00a0a secondary antibody<\/span> obtained from goat anti mouse IgG<\/span>, IgM<\/span>, NS1<\/span>. After adding the two antibody, inoculate for 2 hours and then add the color developing solution after which the ELISA<\/span> assay is inoculated. After 3 days for assay validation wells are infected with DEN V1, DENV2, DENV3, and DENV4 at a multiplicity of infection ranging from 0.01 \u2013 4. After 72 hours the foci forming immunodetection and in situ<\/span> ELISA<\/span> is compared by pearson\u2019s correlation coefficient. A second validation which will act as control and it is done by infecting the wells with DENV4 at a lower multiplicity of infection (MOI) followed by treatment with IFN-\u03b1<\/span> 2A. IFN- \u03b1<\/span> is an antiviral drug used in the treatment of hepatitis C<\/span> infection, and proved to be an effective in vitro replication inhibitor of several pathogenic<\/span> flavivirus<\/span><\/em>, including dengue<\/span>. Based on that, interferon<\/span>-a 2A is used as a reference control. The results of the foci-forming assay, the in situ<\/span> ELISA<\/span> and a commercially available NS1<\/span>, IgG<\/span>, IgM<\/span> antigen<\/span> ELISA<\/span> were compared. IFN-\u03b1<\/span> 2A was then used as a reference control and the IC50<\/span> (concentration that inhibits 50% of virus infection) was determined using a dose response curve for all dengue<\/span> virus serotypes<\/span>.<\/p>\n

With the preparation of control and determination of concentration that inhibits the virus infection, the antiviral activity of any plant or marine seaweed of interest can be determined. The marine seaweed \/ plant sample of interest is screened and a negative and positive control is prepared in the test plates. The ELISA<\/span> kit wells is then added with heptoma cells at a density of 2 X 104 <\/sup>and then infected with different serotypes<\/span> of dengue<\/span> virus at a multiplicity of infection of 4.0, the plates are then incubated for 2 hours at room temperature. The inoculum is then replaced with 200 ml of plant or seaweed extract dilution. After a 72 h incubation period, cells were fixed and ELISA<\/span> is performed as previously described. Data were normalized as % of infection in relation to the controls, where the OD obtained with the non-infected culture were taken as 0% infection, and the one obtained with the non-treated infected culture as 100% infection.<\/p>\n

A disadvantage of this method is that cytotoxicity<\/span> of the antiviral compound cannot be determined by this method, and hence it is advised to perform a cell viable test before screening for antiviral activity using ELISA<\/span> method. The advantage of this method is that antiviral screening against all 4 dengue<\/span> serotypes<\/span> can be performed in the 96 well in situ<\/span> ELISA<\/span> method.<\/p>\n","protected":false},"excerpt":{"rendered":"

Table of Contents I. Introduction \u2022 Dengue as a vector borne disease caused by a flavivirus \u2022 Prevalence of Dengue in tropical and subtropical regions II. Transmission and Life Cycle of Flavivirus \u2022 Vector transmission through mosquitoes \u2022 Attraction of mosquitoes by volatile organic compounds \u2022 Four main serotypes of dengue virus \u2022 Replication of […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"categories":[1],"tags":[11],"class_list":["post-1543","post","type-post","status-publish","format-standard","hentry","category-uncategorized","tag-elisa"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/posts\/1543","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=1543"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/posts\/1543\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=1543"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/categories?post=1543"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/tags?post=1543"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}